We are studying the structural and genetic basis for generating the diversity of neuropeptide/neurotransmitter phenotypes found in the nervous and endocrine systems. This work involves i) analysis of cis-acting elements determining cell-specific expression and regulation of neuropeptide genes and ii) characterization of the components of neuropeptide/neurotransmitter vesicles responsible for packaging of neurotransmitters and neuropeptides in neuroendocrine cells. 1) Positive regulation of enkephalin expression by protein kinase A and calcium, and negative regulation by protein kinase C occurs directly via altered transcription of the enkephalin gene in chromaffin cells. The bovine enkephalin gene is identical to the rat and human genes (for which positive regulation by PMA has been noted). Thus PMA response element can confer both positive and negative regulation as a function of cell type. 2) Primary cultures of bovine chromaffin cells are used to study cell-specific expression of enkephalin, galanin and chromogranin A, via transfection of promoter/reporter gene constructions. Each of these genes is specifically expressed in bovine chromaffin cells, but each has a unique mode of cell-specific regulation. 3) Neuropeptide and secretory protein (chromgranin) biosynthesis are independently regulated in chromaffin cells: biosynthesis of the latter is constitutive rather than regulated in consistent with a role of in constitutive vesicular biogenesis. Studies of the catalysis of secretory vesicle morphogenesis by expression of a chromogranin A expression plasmid in nonendocrine, non-vesicle-containing recipient cells is underway. 4) A cDNA encoding the monamine vesicular transporter (MAT) has been cloned, sequenced and expressed in fibroblastic cells containing a vacuolar-type ATPase.